Proc. NatI. Acad. Sci. USA Vol. 85, pp. 7521-7525, October 1988 Biochemistry Subunit S1 of pertussis toxin: Mapping of the regions essential for ADP-ribosyltransferase activity (erts toxin/ADP-rlbosylatin/subunlt Sl/site-directed mutagenesis) MARIAGRAZIA PIZZA, ANTONELLA BARTOLONI, ANNA PRUGNOLA, SERGIO SILVESTRI, AND RINo RAPPUOLI Sclavo Research Center, Via Fiorentina 1, 53100 Siena, Italy Communicated by A. M. Pappenheimer, July 11, 1988 (receivedfor review April 8, 1988) ABSTRACT The toxicity of pertussis toxin is mediated by related, nontoxic molecules that can be used as vaccines (20- the ADP-ribosyltransferase activity of subunit S1. To under- 22). stand the structure-function relationship of'subunit Si and For diphtheria and Pseudomonas toxins, a series of stud- guide the construction of nontoxic molecules suitable for ies, which include nitrosoguanidine mutagenesis ofthe genes vaccines, we constructed and expressed in Escherichia coli a (23), characterization of the mutant genes (5) and their series of amino-terminal and carboxyl-terminal deletion mu- products (24-26), photoaffinity labeling of the toxins with tants as well as a number of molecules containing amino acid NAD (27, 28), site-directed mutagenesis (29, 30), and crys- substitutions. The shortest peptide still retaining enzymatic tallographic structure of the Pseudomonas exotoxin A (31) activity contains amino-acids 2-179. Within this region we have led to the identification of amino acids essential to the identified three mutants in which amino acid substitutions enzymatic activity of the proteins. For example, Glu-148 of abolish the enzymatic activity. Mutation of amino acids 8 and diphtheria toxin and Glu-553 of Pseudomonas -exotoxin A, 9 or.50 and 53, located within the region of the S1 subunit of located within the catalytic site of the two enzymes, cannot pertussis toxin homologous to cholera toxin, causes loss of be replaced even with an aspartic acid without abolishing enzymatic activity. Outside this homology region, substitution enzymatic activity (29, 30). of Glu-129 with glycine or aspartic acid also eliminates the We used a similar approach to study the structure-function enzymatic activit of the Si subunit. In this respect, Glu-129 relationship ofthe S1 subunit ofpertussis toxin. By carboxyl- resembles the glutamic acid that is crucial for the catalytic and amino-terminal deletion analysis and site-directed mu- activity of diphtheria and Pseudomonas toxins. Once intro- tagenesis, we identified at least three regions of the S1 duced into the Bordetela perussis chromosome, the above subunit that are essential for enzyme function. Substitutions mutations should lead to the synthesis of nontoxic pertussis within these regions produce enzymatically inactive mole- toxin molecules suitable for vaccine production. cules. ADP-ribosylation of the target substrates in eukaryotic cells MATERIALS AND METHODS is a common mechanism of action of many bacterial protein toxins (1-3). The best-studied molecules that adopt this Construction of the Si Deletion Mutants. The S1 subunit of mechanism are diphtheria toxin (4-7), Pseudomonas exotox- pertussis toxin was expressed in Escherichia coli fused to the in A (8-10), cholera toxin (11-13), and pertussis toxin (14- 98 amino-terminal amino acids of the MS2 polymerase. This 16), but several other toxins possessing ADP-ribosyltrans- fusion protein (PTE255) contains amino acids 2-235 ofthe S1 ferase activity have also been described (17, 18). The toxins subunit and is enzymatically active (32). The gene coding for usually contain two functional moieties: A, which is enzy- the amino acids 2-235 is contained within a BamHI-Xba I matically active, and B-, which recognizes and binds the fragment flanked by an EcoRI site at the 5' end and a HindIII receptors on the cell surface facilitating the entry of the site at the 3' end. To obtain the plasmids expressing carboxyl- enzymatically active subunit into the target cells. terminal deletion mutants of the S1 protein, the plasmid The proteins that are ADP-ribosylated by diphtheria, pTE255 was digested first with Xba I and then with Nco I, Pseudomonas, cholera, and pertussis toxins are GTP-binding Nru I, BalI, Sal I, and Sph I, respectively.' The sticky ends proteins involved in protein synthesis (diphtheria and Pseu- generated by the restriction enzymes were then repaired by domonas toxins) or in the transfer of signals through the the large fragment of DNA polymerase, and the plasmids membrane of eukaryotic cells (cholera and pertussis toxins) were circularized by DNA ligase. During this process, the (1, 2). In spite of the common mechanism of action of these natural stop codon of the S1 subunit was lost, and therefore toxins, little or no similarity has been detected by computer the new proteins contained a few amino acids fused at the analysis of their primary structures: only a short amino- carboxyl terminus: NCO, NRU, and SPH proteins had the terminal similarity could be found between fragment A of following carboxyl-terminal unrelated amino acids: Leu-Pro- cholera toxin and the S1 subunit of pertussis toxin (15, 16). Arg-Ala-Phe-Arg. BAL and SAL proteins contained, respec- However, after mapping the functional domains ofdiphtheria tively, 50 and 16 amino acids deriving from the sequence of and Pseudomonas exotoxin A, a significant similarity has pBR322 (33) fused at the carboxyl terminus. To generate the been discovered between the functionally equivalent regions amino terminal deletion mutants, the plasmid pTE255 was cut in the two enzymes (19). with BamHI and then with Sph I, Sal I, and Bal I, respec- A better understanding of the relationship between the tively. The sticky ends were then repaired by the large structure and function of these proteins should help clarify fragment of DNA polymerase, and the fragment was ligated the mechanism of action of the toxins and provide a sound by DNA ligase. By this manipulation, the deleted S1 gene was theoretical basis for the construction of immunologically inserted in the same frame of the MS2 polymerase. Mutants 34A and NCO/BAL were obtained by a similar procedure, the with BamHI/BstNl and The publication costs ofthis article were defrayed in part by page charge cutting plasmid pNCO payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: mAb, monoclonal antibody. 7521 Downloaded by guest on October 2, 2021 7522 Biochemistry: Pizza et al. Proc. Natl. Acad. Sci. USA 85 (1988) BamHI/BalI, respectively. The amino acids of the S1 sub- glutamic acid; 31, TACTCCGTTTTCGTGGTC, which unit, which are expressed by each ofthe deletion mutants, are changed Thr-159 to lysine; 31G, GAATAGGCCGTGGTCG- shown in Fig. 1. The M, values for the fusion proteins are as TG, which changed Glu-160 to glycine; 28D, GCCAGA- follows: NCO, 35,000; NRU, 31,500; BAL, 31,100; SAL, TAGTCGCTCTGG, which changed Glu-129 to aspartic acid; 24,300; SPH, 19,200; 16A, 35,500; 34A, 29,300; SPH/HIND, 1617, the mutations 16 and 17 were combined, and, therefore, 29,200; 255/SAL, 24,700; 255/BAL, 23,100; NCO/BAL, in this mutant Asp-109 and Ala-124 are changed to glycine 20,400; and 18, 20,000. and aspartic acid, respectively. The presence of the unrelated amino acids did not have ADP-Ribosylation. The mutant proteins were assayed for detectable effects on the enzymatic activity (in the case of their ability to ADP-ribosylate transducin as already de- mutants NCO and NRU) or on the stability of the recombi- scribed (32, 36). In particular, after temperature induction of nant molecules; in fact, all ofthem gave only one major band a 10-ml culture, the inclusion bodies were prepared (32), and in sodium dodecyl sulfate/polyacrylamide gel stained with the partially purified fusion proteins were resuspended in 200 Coomassie blue, which, with the exception ofmutant 18, was ,ul of 8 M urea. Five microliters were then used for the recognized by one of the monoclonal antibodies (niAbs) ADP-ribosylation reaction; after activation in a mixture (Table 1). The immunological reactivity of mutant 18 was containing 5 .d ofurea extract, 15 ,ul of water, and 7 /1 of 0.1 confirmed by immunologic blotting by use of a rabbit poly- M dithiothreitol for 30 min, 5 pl of 2 M Tris, pH 7.5, 1 td of clonal antiserum against a synthetic peptide covering the 100 mM ATP, 1 t4 of 10 mM GTP, 10 !d of0.25 M thymidine, region 172-194. The folding of the recombinant molecules 10 ,ul of retinal outer segment membranes (ROS), and 80 p1 also seemed to be unaffected because after in vitro refolding, ofH20 were added. The reaction mixture was then incubated the truncated fragments could generate a conformational at room temperature for 2 hr and centrifuged; the insoluble epitope that requires the interaction of the amino- and pellet containing the transducin was solubilized in a sodium carboxyl-terminal parts of the molecule (34). dodecyl sulfate-loading buffer and was loaded in a 12.5% Site-Directed Mutagenesis. To mutagenize the S1 gene, the acrylamide gel. After electrophoresis, the labeled transducin restriction fragment BamHI-Xba I ofplasmid pTE255, which was visualized by autoradiography. For quantitative analy- contains the S1 gene, was subcloned in Bluescript-KS (Stra- sis, the bands containing transducin were cut out, and tagene, San Diego, CA), and the single-stranded DNA was radioactivity was counted in a 13 counter. mutagenized using oligonucleotide primers as described (35). NAD+ Glycohydrolase Activity. The samples in 8 M urea After mutagenesis, the BamHI-Xba I fragment was sub- were prepared as for the ADP-ribosylation reaction. Then, 5 cloned again into the expression vector pEX34b (32) for the 1d were diluted in the reaction mixture and incubated for 18 expression of the mutant protein.